The research goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the human erythrocyte ankyrin gene. Erythrocyte ankyrin, an important constituent of the erythrocyte membrane skeleton, is the prototype of a family of proteins that couple integral membrane proteins to the spectrin cytoskeleton. the studies in this proposal include identification and analysis of cis-acting sequences and trans-acting factors regulating tissue-specific expression of the erythrocyte ankyrin gene. Comparison of these regulatory elements with those of other erythroid genes will aid in the elucidation of the molecular mechanisms responsible for the tissue-specific expression of ankyrin. Another specific aim of this proposal is the study of the molecular basis and functional significance of the large number of different ankyrin isoforms generated by alternative splicing of the gene transcript. A third specific aim is the analysis of the somewhat unexpected expression of erythrocyte ankyrin gene in brain. Finally, the knowledge obtained from these studies will be applied to the identification of molecular defects of ankyrin in hereditary spherocytosis, an important cause of inherited hemolytic anemia. The general methodology to be utilized in this research includes: cloning and structural analysis of the cDNA and genomic DNA segments of the human erythrocyte ankyrin gene relevant to its expression and regulation by use of recombinant DNA technology; study of cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-I footprinting, methylation interference techniques and site-directed mutagenesis followed by in vivo and in vitro analyses; and study of cDNA and genomic DNA from individuals with ankyrin-associated hereditary spherocytosis by use of the polymerase chain reaction (PCR) technique. This laboratory research will be performed by the primary investigator, Dr. Patrick G. Gallagher, in conjunction with a didactic program under the guidance of a sponsor, Dr. Bernard G. Forget, and an advisory committee. This proposal will allow the candidate to develop the technical expertise, fundamental knowledge and intellectual scientific approach necessary for an independent and productive career in investigative molecular genetics and its application to the study of inherited disorders.